Optimization of ultrasonic extraction of polysaccharides from dried longan

Optimization of ultrasonic extraction of polysaccharides from dried longan pulp using response surface methodologyKui Zhong a,b ,Qiang Wang a,*a Institute of Agro-food Science and Technology,Chinese Academy of Agricultural Science,Beijing 100094,China bCollege of Food Science &Technology,Huazhong Agricultural University,Wuhan,Hubei Province 430070,Chinaa r t i c l e i n f o Article history:Received 24September 2009Received in revised form 19October 2009Accepted 26October 2009Available online 30October 2009Keywords:Ultrasonic extraction Longan polysaccharides Dried longan pulpResponse surface methodology (RSM)a b s t r a c tUltrasonic technology was applied for polysaccharides extraction from the dried longan pulp and response surface methodology (RSM)was used to optimize the effects of processing parameters on poly-saccharides yields.Three independent variables were ultrasonic power (X 1),extraction time (X 2)and ratio of water to raw material (X 3),respectively.The statistical analysis indicated that three variables and the quadratic of X 1and X 2had significant effects on the yields,and followed by the significant interaction effects between the variables of X 2and X 3(p <.05).A mathematical model with high determination coef-ficient was gained and could be employed to optimize polysaccharides extraction.The optimal extraction conditions of polysaccharides were determined as follows:Ultrasonic power 680W,extraction time 4.5min,ratio of water to raw material 25mL/g.Under these conditions,the experimental yield of poly-saccharides was 4.455±0.093%,which was agreed closely with the predicted value (4.469%).Crown Copyright Ó2009Published by Elsevier Ltd.All rights reserved.1.IntroductionLongan (Dimocarpus Longan Lour.)is an important tropical fruit in Southeast Asia,such as China,Vietnam and Thailand.It’s taste,nutritional and favored by many consumers in the world.Longan has also been used as a traditional chinese medicine since ancient times,and great attentions have been paid for their great health effects (Yang,Zhao,Shi,Yang,&Jiang,2008a ),such as promoting blood metabolism,soothe nerves,relieve insomnia,etc.(Yang et al.,2008b ).Polysaccharides and lignin in the Longan fruit have been considered the main functional compositions for these health effects (Yang,Jiang,Wang,Zhao,&Sun,2009).Polysaccharides from plant,epiphyte and animals extracts are an interesting source of additives for several industries,in particu-lar food and drug industry (Forabosco et al.,2006).They play important roles in the growth and development of living organ-isms,and have been widely studied in recent years due to their un-ique biological,chemical and physical properties (Schepetkin &Quinn,2006).Whereas,there have been only a few reports on lon-gan polysaccharides and few on its functional effects.One of the reasons is the lack of high efficient extraction technology of poly-saccharides from Longan pulp.Hot-water technology is the main extraction method of longan polysaccharides in recent research,which is a classical extraction of polysaccharides.It usually re-quires long extraction time,high temperature and extraction effi-ciency is low (Li,Ding,&Ding,2007).Therefore,it’s essential and desirable to find an economical and high efficient extraction meth-od of polysaccharides from longan fruit.Ultrasonic treatment has been employed for preparing polysac-charides from different plant materials in recent years and showed the great extraction efficiency (Hemwimon,Pavasant,&Shotipruk,2007;Hofmann,Kappler,&Posten,2006;Hromadkova &Ebringer-ova,2003;Hromadkova,Ebringerova,&Valachovic,1999;Wang,Cheng,Mao,Fan,&Wu,2009).This great extraction efficiency by ultrasonic treatment is mainly attributed to its mechanical effects,which greatly facilitate mass transfer between immiscible phases through a super agitation (Vinatoru et al.,1997),and the most important mechanical effects of ultrasonic treatments are microjetting and microstreaming (Tsochatzidis,Guiraud,Wilhelm,&Delmas,2001;Velickovic,Milenovic,Ristic,&Veljkovic,2006).Response surface methodology (RSM)is an affective statistical technique for optimizing complex processes.The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions.Therefore,it is less laborious and time-consuming than other approaches re-quired to optimize a process (Giovanni,1983).It is wide used in optimizing the extraction process variables,such as polysaccha-rides,anthocyanins,vitamin E,phenolic compounds and protein from varied materials (Cacace &Mazza,2003;Chandrika &Fereid-oon,2005;Ge,Ni,Yan,Chen,&Cai,2002;Lee,Kim,&Kwon,2005;Li &Fu,2005;Liyana-Pathirana &Shahidi,2005a;Qiao et al.,2009).Box–Behnken design (BBD),one of RSM,only have three levels,and need fewer experiments.It’s more efficient and easier to arrange0144-8617/$-see front matter Crown Copyright Ó2009Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.carbpol.2009.10.066*Corresponding author.Tel./fax:+861062815837.E-mail address:caaswangqiang@ (Q.Wang).Carbohydrate Polymers 80(2010)19–25Contents lists available at ScienceDirectCarbohydrate Polymersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p oland interpret experiments in comparison with others and widely used by many researches(Box&Behnken,1960;Ferreira et al., 2007).In this study,the main objective was to optimize ultrasonic technology conditions for the extraction of polysaccharides from dried Longan pulp.RSM was designed to systemic analyze the ef-fects of extraction parameters on the yields of polysaccharides from dried Longan pulp and their interactions.2.Materials and methods2.1.Experimental materials and chemicalsDried longan(D.longan Lour.)fruit was purchased in a local commercial market and producing area was Putian,Fujian Prov-ince,China.The dried longan fruit was peeled,seeded and air-dried at50°C for balancing the water.Then,the dried longan pulp was grinded by a miller(A11basic,ZKAÒ-WERKE,Germany),collected and stored in desiccator at room temperature(15–20°C)until used (less than one month).All chemicals used in this investigation were analytical grade and purchased from Beijing Chemicals Co. (Beijing,China).2.2.Extraction of polysaccharides from dried Longan pulp with ultrasonic treatmentThe process of polysaccharides extraction from dried longan pulp by ultrasonic treatment was performed in an ultrasonic cell disintegrator(JY92-II,Xinzhi Bio-technology and Science Inc.,Ling-bo,ZheJiang Province,China).Two grams of dried longan pulp powders were extracted with distilled water in a100-mL beaker, then the beaker was held in the ultrasonic cell disintegrator and exposed to extract for different time at varied ultrasonic power. Ice bathing was used to ensure the temperature of solution was be-low50°C in the whole extraction processing.2.3.Isolation and determination yield of Longan polysaccharidesAfter the extraction with ultrasonic treatment,the extracted slurry was centrifuged at4200rpm/min for20min to collect the supernatant,and the insoluble residue was treated again for2–3 times as mentioned above.The supernatant was incorporated and concentrated to one-fifth of initial volume using a rotary evap-orator(Senco Technology and Science Inc.,Shanghai,China)at 55°C under vacuum.The resulting solution was mixed with four volumes of dehydrated ethanol(ethanolfinal concentration,80%) and kept overnight at4°C.Then the solution was centrifuged at 4200rpm/min for20min,washed six times with dehydrated eth-anol,and the precipitate was collected as crude extract.The extract was air-dried at50°C until its weight was constant,and then was weighted with a balance(AY120,SHIMADZU,Japan).The percent-age polysaccharides yield(%)is calculated as follows:Yieldð%Þ¼weight of dried crude extractionðgÞweight of longan pulp powderðgÞÂ100ð1Þ2.4.Experimental designA three level,three variable Box–Behnken factorial design(BBD) (Design Expert software,Trial Version6.0.5,Stat-Ease Inc.,Minne-apolis,MN)was applied to determine the best combination of extraction variables for the yields of longan polysaccharides.Three extraction variables considered for this research were X1(ultra-sonic power),X2(extraction time),and X3(ratio of water to raw material)(Li et al.,2007),and the proper range of three variables were determined on the basis of single-factor experiment for the polysaccharides production(Table1).Table1listed the whole de-sign consisted of17experimental points,five replicates(treatment 13–17)at the centre of the design were used to allow for estima-tion of a pure error sum of squares.The triplicates were performed at all design points in randomized order.Experimental data werefitted to a quadratic polynomial model and regression coefficients obtained.The non-linear computer-generated quadratic model used in the response surface was as follows:Y¼b00þX4i¼0b i X i0þX4i¼0b ii X2i0þX4i¼0X4j¼0b ij X i X jð2Þwhere Y is the measured response associated with each factor lever combination;b0is an intercept;b i is regression coefficients com-puted from the observed experimental values of Y;and X i is the coded levels of independent variables.The terms X i X j and X i2repre-sent the interaction and quadratic terms,respectively.2.5.Statistical analysesData were expressed as means standard errors(SE)of three rep-licated determinations.The responses obtained from each set of experimental design(Table1)were subjected to multiple non-lin-ear regressions using the Design Expert software(Trial Version 6.0.5,Stat-Ease Inc.,Minneapolis,MN).The quality of thefit of the polynomial model equation was expressed by the coefficient of determination R2,and the significances of the regression coeffi-cient were checked by F-test and p-value.3.Results and discussion3.1.Fitting the modelA regression analysis(Table2)was carried out tofit mathemat-ical models to the experimental data aiming at an optimal region for the responses studied.Predicted response Y for the yield of Lon-gan polysaccharides could be expressed by the following second-order polynomial equation in terms of coded values:Y¼4:26þ0:65X1þ0:096X2À0:040X3À0:67X21À0:11X22þ6:250Â10À4X32À0:022X1X2À0:015X1X3À0:068X2X3ð3ÞTable1Box–Behnken experimental design with the independent variables.Run Coded variable levels Yield of polysaccharide(%)X1X2X3Actual values Predicted values 1À1À10 2.750 2.72021À10 4.039 4.0563À110 2.974 2.9574110 4.175 4.2055À101 2.945 2.979610À1 4.315 4.3017À101 2.915 2.9298101 4.225 4.19190À1À1 4.030 4.0261001À1 4.372 4.356110À11 4.066 4.08312011 4.135 4.13913000 4.340 4.26514000 4.251 4.26515000 4.243 4.26516000 4.270 4.26517000 4.221 4.26520K.Zhong,Q.Wang/Carbohydrate Polymers80(2010)19–25where Y is the yield of longan polysaccharides(g),and X1,X2,and X3 are the coded variables for ultrasonic power,extraction time and the ratio of water to the raw material,respectively.In general,exploration and optimization of afitted response surface may produce poor or misleading results,unless the model exhibits a goodfit,which makes checking of the model adequacy essential(Liyana-Pathirana&Shahidi,2005b).The F-ratio in this table is the ratio of the mean square error to the pure error ob-tained from the replicates at the design centre.The significance of the F-value depends on the number of degrees of freedom(DF) in the model,and is shown in the p-value column(95%confidence level).Thus,the effects lower than.05in this column are signifi-cant(Cai,Gu,&Tang,2008;Qiao et al.,2009).Table2listed the analysis of variance(ANOVA)for thefitted quadratic polynomial model of extraction yields of longan polysac-charides.F-test suggested that model had a very high model F-va-lue(F=302.64)and a very low p-value(p<.0001),indicating this model was highly significant.The lack offit measures the failure of the model to represent the data in the experimental domain at points which are not included in the regression.As showed in Table 2,F-value and p-value of the lack offit were.91and.5103,respec-tively,which implied it was not significant relative to the pure er-ror and indicated that the model equation was adequate for predicting the yield of longan polysaccharides under any combina-tion of values of the variables.R2adj(adjusted determination coef-ficient)is the correlation measure for testing the goodness-of-fit of the regression equation.Higher it is the better degree of correla-tion between the observed and predicted values(Ravikumar, Ramalingam,Krishnan,&Balu,2006).The value of R2adj for Eq.(3)was.994,which was reasonably close to1and implied that only less1.0%of the total variations were not explained by model. Meanwhile,it also confirmed that the model was highly significant and indicated a high degree of correlation between the observed and predicted data.Coefficient of variation(CV)indicates the de-gree of precision with which the experiments are compared.A rel-atively low value of CV(1.14)in Table2,which showed a better precision and reliability of the experiments carried out.The significance of each coefficient was determined using p-va-lue in Table3.The p-value is used as a tool to check the significance of each coefficient and the interaction strength between each inde-pendent variable(Muralidhar,Chirumamilla,Ramachandran,Mar-chant,&Nigam,2001).The corresponding variables would be more significant at greater F-value and smaller p-value(Atkinson&Do-nev,1992).The data in the Table3indicated that all the indepen-dent variables(X1,X2,X3)and two quadratic terms(X12and X22) significantly affected the yield of longan polysaccharides,and there was significant interaction between extraction time(X2)and ratio of water to raw material(X3).Meanwhile,the ultrasonic power (X2)was the major factor affecting the yield of polysaccharides. 3.2.Analysis of response surfaceThe3D response surface and2D contour plots are the graphical representations of regression equation.They provide a method to visualize the relationship between responses and experimental levels of each variable and the type of interactions between two test variables.The shapes of the contour plots,circular or elliptical, indicate whether the mutual interactions between the variables are significant or not.Circular contour plot indicates that the inter-actions between the corresponding variables are negligible,while elliptical contour plot indicates that the interactions between the corresponding variables are significant(Muralidhar et al.,2001). The relationship between independent and dependent variables was illustrated in tri-dimensional representation of the response surfaces and two-dimensional contour plots generated by the model for yield of polysaccharides(Figs.1–3),two variables were depicted in one tri-dimensional surface plots while the other vari-able kept at level zero.It is clear that the yield of polysaccharides was sensitive to minor alterations of the test variables(ultrasonic power,extraction time and ratio of water to raw material).The interaction relationships of ultrasonic power(X1)with the extraction time(X2)and radio of water to material(X3)on the yield of polysaccharides were shown in Figs.1and2,respectively,and which indicated these three variables all had significant effect on the yield of longyan polysaccharides.As shown in Figs.1and2, the ultrasonic power(X1)and extraction time(X2)had positive im-pact on the polysaccharides production,while the yield changed slightly when the ratio of water to material(X3)was in the range of20mL/g to50mL/g.Yield of polysaccharides rapid enhanced with the increasing of ultrasonic power(X1)and reached to the peak value at668.43W.With the farther increasing of ultrasonic power(X1),the yield went to slight decrease.Longer extraction time(X2)had positive effects on the yield extraction,and had a critical value at 4.6min when at a constant ultrasonic power (668.43W).This suggested more yield was resulted at higher ultrasonic power,longer extraction time and lower ratio of water to material.It was considered higher extraction efficiency of polysaccha-rides at higher ultrasonic power due to the increase in the number of cavitation bubbles formed and enhance mass transfer rates. However,less yield was resulted at farther increasing ultrasonic power.The same result was gained by Li et al.(2007),and was con-sidered for that a part of polysaccharides could be more depoly-merize into some free sugars.It was shown that the interactions between the ultrasonic power and other two extraction variables did not impact the yield of polysaccharides significantly(Table3,Figs.1and2),in spit of the ultrasonic power was the major factor affecting the yield of polysaccharides.This observation was in agreement with previous investigation.Li et al.(2007)researched the optimization of the ultrasonically assisted extraction of polysaccharides from Zizyphus jujuba cv.Jinsixiaozao by RSM,and analysed the effects of interac-tions of extraction variables on the yield of polysaccharides.They also indicated that the interactions between ultrasonic power and extraction time,and ultrasonic power and ratio of water toTable2Analysis of variance for thefitted quadratic polynomial model of extraction ofpolysaccharides.Source SS DF MS F-valueProb>FModel 5.4290.60302.64<0.0001Residual0.0147 1.989Â10À3Lack offit 5.658Â10À33 1.886Â10À3Pureerror8.266Â10À34 2.066Â10À30.910.5103Cor Total 5.4316R2=0.997R2Adj=0.994CV=1.14Table3Estimated regression model of relationship between response variables(yield ofLongan polysaccharides)and independent variables(X1,X2,X3).Variables DF SS MS F-value p-ValueX11 3.340 3.3401679.70<.0001X210.0740.07437.360.0005X310.0130.013 6.480.0384X1X11 1.870 1.870937.860<.0001X2X210.0560.05627.930.0011X3X31 1.645Â10À6 1.645Â10À68.269Â10À4.9779X1X21 1.936Â10À3 1.936Â10À30.970.3567X1X319.000Â10À49.000Â10À40.450.5227X2X310.0190.0199.370.0183K.Zhong,Q.Wang/Carbohydrate Polymers80(2010)19–2521material caused no significant effect on the extraction yield,whileultrasonic power had significant effect.Fig.3showed the response surface plot at various extraction times (X 2)and ratio of water to material (X 3).The response curves demonstrated that higher yield at longer extraction time.The re-sponse curves were comparatively smooth at lower extraction time,indicating the less effect on the increasing of the yields extraction when ratio of water to material changed in the range from 20mL/g to 50mL/g.However,the yield decreased with the farther enhancing of ratio of water to material at longer extraction time.This result indicated that extraction time (X 2)had a different extent of influence on extraction yield in different ratio of water to3.006143.292413.5786822K.Zhong,Q.Wang /Carbohydrate Polymers 80(2010)19–25materials(X3),and significant interactions were existed between extraction time(X2)and ratio of water to raw material(X3).Higher yields of polysaccharides were resulted at longer extraction time and lower ratio of water to material in the experimental range.As shown in Fig.3and Table3,the interactions of extraction time and ratio of water to materials had significant effect on the extraction yields,which was the same with other research results (Rodrigues,Pinto,&Fernandes,2008;Wang et al.,2009).This con-3.292413.578684.3488K.Zhong,Q.Wang/Carbohydrate Polymers80(2010)19–2523clusion was inconsistent with the observation obtained by Li et al. (2007),who reported that this interaction caused no significant ef-fect on the extraction yields at a constant ultrasonic power.This contradiction was possibly due to the large difference in parame-ters of sonic power.In this study,the sonic power(>400W)were far greater than that(60W)in Li et al.(2007).3.3.Optimization of extracting parameters and validation of the modelThe suitability of the model equation for predicting the opti-mum response values was tested using the selected optimal condi-tions.The maximum predicted yield and experimental yield of longan polysaccharides were given in Table4.Additional experi-4.1512224K.Zhong,Q.Wang/Carbohydrate Polymers80(2010)19–25ments using the predicted optimum conditions for polysaccharides extraction were carried out:Ultrasonic power of668.43W,extrac-tion time of4.6min,ratio of water to material24.25mL/g,and the model predicted a maximum response of4.469%.To ensure the predicted result was not bias the practical value,experiment rechecking was performed using this modified optimal conditions: Ultrasonic power of680W,extraction time of4.5min,ratio of water to material25mL/g.A mean value of 4.456±0.063% (N=5)was gained,which was in agreement with the predicted va-lue significantly(p>.05),obtained from real experiments,demon-strated the validation of the RSM model.The results of analysis confirmed that the response model was adequate for reflecting the expected optimization(Table4),and the model of Eq.(3) was satisfactory and accurate.4.ConclusionUltrasonic technology was performed for the polysaccharides extraction from dried longan pulp in order to increase the yield extraction.Based on the single-factor experiments,Response sur-face methodology(RSM)was used to estimate and optimize the experimental variables-ultrasonic power(W),extraction time (min)and ratio of water to raw material(mL/g).All the indepen-dent variables,quadratic of ultrasonic power and extraction time had high significant effects on the response values,followed by the significant interaction effects between the extraction time and ratio of water to material.A high correlation of the quadratic polynomial mathematical model was gained and could be great employed to optimize polysaccharides extraction from Longan by ultrasonic technology.The optimal extraction conditions for the polysaccharides were determined as follows:Ultrasonic power 680W,extraction time4.5min,ratio of water to material25mL/g. 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